摘要
将鸡传染性支气管炎病毒（ Ｉ Ｂ Ｖ） Ｓ１ 基因 ｃ Ｄ Ｎ Ａ 克隆至含有起始密码 Ａ Ｔ Ｇ 的转移载体 ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４，构建成重组转移质粒 ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１． Ｈｏｌｔｅ，再与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ， ｇａｌ＋ ）共转染 草地夜蛾（ Ｓｆ９） 细胞， 经空斑纯化得 到已插入 Ｓ１ 基 因并能形成多角体 的重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。以重组毒株感染 Ｔｎ５ Ｂ１ 细胞，在不同时间收取感染了病毒的细胞进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗ ｅｓｔｅｒｎ 印迹检测。 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 基因，表达产物为约 １００ ０００ 的融合蛋白，与预计的表达修饰后的产物大小相符，推测此蛋白已糖基化。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ凝胶薄层色谱分析结果显示，感染病毒后 ４８、７２、９６ ｈ， Ｓ１ 蛋白分别占细胞内总蛋白量的 ２８７％ 、３５８％ 、３７１％ 。
The S1 region of the avian infectious bronchitis virus(IBV) S protein is responsible for inducing VN and HI antibodies. Insertion of the cDNA of S1 gene of IBV, Holte strain into transfer vector plasmid pSXIVVI +X3/4 by directional cloning was done. The resulting recombinant transfer plasmid pSXIVVI +X3/4 S1.Holte and the parent virus(TnNPV SVI G) DNA were used to co transfect Spodotera frugiperda(Sf) cells and recombinant virus TnNPV (X3/4)S1.Holte OCC +, which included S1 gene and could form polyhedra, were plaque purified. The expression product of S1 gene with a molecular weight of 10 5 was detected by SDS PAGE and Western blot in Tn 5B1 cells infected with TnNPV (X3/4)S1.Holte OCC +. The expression product wound be a completely N glycosylated fusion protein. The expression product levels in Tn 5B1 cells after 48, 72 and 96 h postinfection, were 28.7%, 35.8% and 37.1% by SDS PAGE gel thin layer chromatography.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1999年第5期421-424,共4页
Chinese Journal of Veterinary Science
基金
国家教委高校博士点基金
